Project Summary Identifying maintenance, amplifying and overwintering hosts is one of critical factors in understanding and ultimately controlling vector-borne diseases. There is considerable evidence from the literature and our own preliminary studies that reptiles and amphibians infected with various arboviruses develop a viremia of sufficient magnitude to likely infect mosquitoes. Further, and especially when environmental temperature is reduced, viremia in these ectothermic vertebrates can persist for weeks or months, suggesting a potential role in overwintering. Despite these intriguing hints of the importance of ectothermic vertebrates in the ecology of arbovirus infections, we lack a broad understanding of the pathogenesis of such host-virus interactions, particularly under varying environmental conditions. The recent emergence in the New World of chikungunya and Zika viruses provides further impetus for an expanded evaluation of non-conventional hosts for arboviruses. Our long-term objective is to determine whether reptiles and amphibians are important in natural arboviral transmission cycles, but realizing that goal will be logistically difficult without a much better understanding of which virus-hosts are worth evaluating. Our approach is based on first testing an array of diverse arboviruses for their ability to infect and induce viremia in multiple ectothermic species in order to better define the possibilities for specific virus-host interactions. In conjunction with these exploratory studies, we will expand upon preliminary studies to characterize the influence of low temperature on duration and recrudescence of viremia for Zika and two other viruses in selected reptiles and amphibians. Finally, we will exploit an artificial ecosystem to address the ultimate question of whether reptiles and amphibians can actually serve as hosts important to arbovirus transmission cycles. Collectively, these studies will greatly enhance our understanding of the potential importance ectothermic hosts in arbovirus transmission and set the stage for future studies to evaluate such interactions in the natural world.